The enzymes, if soluble in water or in a mixture of water and other co-solvent, can be used for a single conversion cycle. To overcome this drawback, the technique of immobilization has been developed.
The increasing use of enzymes as catalysts in industrial processes has led to increasing demand of enzymes in immobilized form. The immobilized enzymes frequently termed as ‘biocatalysts’ are widely used for industrial organic synthesis and biotransformation. According to a 2002 review, there are more than 130 biotransformation currently performed in an industrial scale using biocatalyst, mostly in the manufacture of pharmaceutical intermediates and other fine chemicals. Over the years this widely accepted technique of immobilization, has witnessed significant changes and improvement. Today the immobilizations are directed to specific use and tailor made for purpose of use like immobilized antibodies, macromolecular complexes and structural proteins. Immobilized enzymes (biocatalyst) however command greater attention.
The biocatalysts can be produced using whole living or dead cells or the enzymes made thereof. The source of enzyme may vary from plants and animals to microorganisms. The expanded pool of enzymes and advances in protein engineering has made it possible to produce economically viable biocatalyst on a commercial scale.
The enzymatic reactions are highly specific which is based on specific properties of the enzymes such as substrate specificity, activity, enantio-selectivity, productivity, stability, pH and temperature optimum profiles and so forth. The commercialization of many enzymes is hampered by lack of operational stability coupled with relatively high price. This impediment can be overcome if one can find an effective method for immobilization. If successful, this not only results in improved selective properties, operational stability but allows for the facile separation, reuse of enzyme and simplifies down stream processing. Hence to develop a suitable biocatalyst the selection of suitable immobilization process becomes crucial.
Conceptually, immobilizations have several approaches. Amongst them are adsorption, carrier binding, entrapment, and cross linking. Physical adsorption relies on the affinity of enzyme with the support. Though simple, the interaction is weak and the enzyme can be readily desorbed and lost. Carrier binding involves connecting the enzyme to a water insoluble support by ionic or covalent bonds. This process has a disadvantage of partial inactivation of the enzyme molecule because of strong chemical bond. Also the target enzymes are required to be in more purified form which eventually increases the cost of catalyst production.
The third method of immobilization involves entrapment of a given enzyme in gel or microcapsules made of suitable organic or inorganic natural or synthetic polymers. This method has an advantage as it does not entail protein inactivation due to strong covalent bond modification. The formation of pores of a given size of the selected gel matrix or fibers can be suitably adjusted so as to hold up high molecular weight enzyme molecules. And yet permit selective passage of small reactant molecules and consequently, preclude the otherwise possible separation of the enzyme from the carrier. More often the entrapment method is combined with cross linking method in a number of configurations. Specific bi-functional agents like glutaraldehyde which form covalent bonds with the protein molecule resulting in formation of large aggregates. Cross linking also imparts rigidity and physical strength to the entrapped enzyme. In addition, supports generally used in entrapment seem to be compatible with whole cells and enzymes with low specificity and lower purity as compared to those used in covalent binding. This essentially makes the process more cost effective.
Ample Literature is available on use of different materials and products of immobilized enzymes formed thereof.
An article on “Immobilization of permeabilized whole cell penicillin G acylase from Alcaligenes faecalis using pore matrix cross linked with glutaraldehyde” by Cheng, Shiwei et al, (Biotechnology Letters, Volume 28, Number 14, July 2006, pages 1129-1133) discloses the activity of penicillin G acylase from Alcaligenes faecalis increased 7.5-fold when cells were permeabilized with 0.3% (w/v) CTAB. The treated cells were entrapped by polyvinyl alcohol crosslinked with boric acid, and crosslinked with 2% (v/v) glutaraldehyde to increase the stability. The conversion yield of penicillin G to 6-aminopenicillanic acid is 75% by immobilized system in batch reaction. No activity is lost after 15 cycles and about 65% enzyme activity is retained at the end of the 31st cycle.
U.S. Pat. No. 4,727,030 and U.S. Pat. No. 4,978,619 also reports use of gel matrix for immobilization of enzymes or cells.
Recently, Cross linked Enzyme Aggregates (CLEAs) as immobilized enzymes, has gained much importance and is looked upon as lucrative alternative to use of polymers for immobilization. However, its industrial viability needs to be further evaluated as the CLEA particles of most of the enzyme is too small and poses serious filtration problems. Though, CLEA offers high advantage of very high specific activity of enzyme, this operational issue of filtration impediments commercial exploitation.
Significant work has been reported and published in this particular field but few techniques have been successful at commercial level. Penicillin acylases of microbial origin are currently used in immobilized form for the synthesis of intermediates of beta lactam antibiotics. Literature reports whole cell penicillin acylase catalyst, enzyme immobilized by entrapment or covalently linked purified enzyme forms, including catalysts based on Cross Linked Enzyme Crystals (CLEC) and Cross Linked Enzyme Aggregates CLEA.
Penicillin acylase immobilized on cross linked p-xylene diamine with glutaraldehyde (Poly-XDA-GA-PA), assemblase(r) 7500 entrapped in Gelatin and Chitosan, PGA-450 FERMASE PA 250, whole cell cross linking on Polyethleneimine supports, are to name a few.
U.S. Pat. No. 5,846,762 claims use of gelatin with propylene glycol & alginate with glutaraldehyde to form gel beads containing entrapped enzyme of different enzymes.
In U.S. Pat. No. 6,060,268 claims preparation of an immobilized penicillin G acylase by covalent bonding to a crosslinked gelled gelatin and other polymers such as alginate chitosan or polyethylene imine.
By whichever method immobilized enzyme is prepared, an ideal industrial immobilized enzyme has to meet several criteria, such as recyclability, broad applicability, cost effectiveness, environmental and safety issues. Further, the conditions on an industrial scale are more severe for immobilizations as there are problems of inactivation of cells or enzymes due to polyfunctional reagents used. Also the low physical strength of immobilized biomaterial may clog the pores leading to diffusional limitations and hence limiting long time catalyst usage. Consequently, the disposal of deactivated immobilized enzymes also has to be accounted for an industrial scale. This is especially evident in case of certain enzymes like immobilized penicillin acylase which are used in tons for production of semisynthetic beta-lactam antibiotics, both in the preparation of intermediates as well as in final drug molecules.
Therefore, there is a need in the art to develop a biocatalyst having high activity and operational stability with multiple usage in hydrolytic and synthetic biocatalyses namely in the area of biotransformation of semisynthetic beta-lactam antibiotics, which the present inventors have achieved in this invention.